Bioaffinity chromatography involves the separation of specific molecules or bioaffinants from solutions through attraction to and binding by specific molecular recognition sites on stationary bioaffinity ligands attached to a chromatographic support.
The chromatographic supports used in bioaffinity chromatography are generally made in the form of beads that are made from polysaccharides, such as agarose and dextran, and other polymers, such as polyethylene oxide. An ideal solid support for use in affinity chromatography should allow for high flow rates with minimal pressure drop when processing in a column configuration. Agarose and dextran supports must be cross-linked (i.e., with epichlorohydrin) in order to achieve even a modest degree of rigidity and stability at low to moderate flow rates (e.g., 10 to 15 cm/min through a 1 cm diameter column with a 15 cm bed height). Much higher flow rates are required. This is because many of the compounds to be separated or purified are present at very low concentrations, and large volumes must flow through a column to obtain useful results.
The beads are generally small, having diameters less than about 250 microns. The widespread use of small beaded supports for column based immunoaffinity protein purification has been based upon two theories. The first theory states that antibodies are large molecules with specific activity at one end and crouding of these molecules reduces their specific activity. The second theory states that for proteins to interact with the majority of the active sites in a bead, it must diffuse into the bead to the location of the site. Thus, since the diffusion rates of most proteins are very slow, the diffusion length and therefore the bead size must be kept small. The correlary to this is that even if most of the antibody is immobilized near the surface of the bead, small beads have a larger surface area to volume ratio than large beads and therefore small beads will have lower local antibody concentrations (less crowding) than large beads for the same bulk antibody concentration. This has lead to the understanding that small beads are the only logical support for column based immunoaffinity protein separations.
The high flow rates necessary to treat large volumes tend to crush prior art beads made from materials such as crosslinked agarose or dextran. Thus, beads used as bioaffinity chromatography solid supports should have high mechanical strength to avoid crushing at high flow rates (e.g., above 20 cm/min in a 15 cm column). It has also been noted that packed column beds of nonspherical or irregular shaped particles tend to compact more readily than columns of spherical particles of the same grade. This makes it desirable to have a solid support made from spherical beads of high mechanical strength.
Certain chromatographic support materials form a gel due to hydration of the solids. These gels are preferred as bioaffinity chromatography stationary phase supports because of the large amount of binding sites available per volume of gel. The high density of binding sites provides great reactive or separative capacity. Gels are characteristically high in liquid content, containing a relatively low percentage of solids, and have a highly porous structure. The highly porous structure enables liquid to flow (perfuse) through, rather than around, the particles forming the gel. However, not all of the internal volume of the gel is necessarily accessible to all species dissolved in the liquid medium flowing through the gel. Thus, molecular sieving can occur as a result of this limited accessibility to species of different molecular size. In fact, this lack of accessibility to the internal gel particle matrices is used in gel permeation chromatography to separate molecules of different sizes.
However, in order for materials to flow through a gel, the matrix must be sufficiently porous to allow access to the internal volume of the particles making up the gel. If the gel is not sufficiently porous, undesirable clogging, molecular sieving, or low flow rates result (note, the term high porosity is used herein to indicate that the internal matrices of the gel particles, or beads, are accessible to molecules of high molecular weight, e.g., &gt;50,000 daltons).
Bead cellulose forms a gel with polar solvents, such as water; and has been recommended as a support for affinity chromatography. This is due to the physical properties of cellulose beads which tolerate high column flow rates. This mechanical strength is achieved without chemical cross-linking. In addition, cellulose is less susceptible to dissolution from a wider range of chromatography solvents than other prior art support materials. Further, cellulose bead matrices do not tend to nonspecifically bind or attract (sorb) species in solution.
Binding interactions between a chromatographic support of cellulose and the components of a solution tend to occur between the ligands bound to the cellulose and the specific solution components with which they possess affinity or avidity. This results in a highly specific chromatographic support. Cellulose is also much cheaper to obtain than other prior art bead materials, such as agarose or dextran, and it is readily available throughout the world. In fact, lignocellulose (cellulose combined with lignin) is even easier to obtain since plants and trees contain lignocellulose, rather than pure cellulose. If beads for bioaffinity chromatography can be made from cellulose or lignocellulose, the efficiency of the method will improve, while the cost will decrease.
Cellulose beads have been successfully used as a support for bioaffinity chromatography of low molecular weight species. For example, Kucera, in "Affinity Chromatography Of Chymotrypsin (E.C. 3.4.21.1) On The Potato Trypsin Inhibitor Bound To Bead Cellulose By The Benzoquinone Method," Journal of Chromatography, 213, (1981) 352-354, herein incorporated by reference, bound trypsin inhibitor, prepared from potatoes, to bead cellulose in an amount of 200 mg per gram of the support. The trypsin inhibitor was bound to the bead cellulose after first activating the cellulose with benzoquinone. Affinity chromatography was then performed on a column having bound trypsin inhibitor using a solution of chymotrypsin in 0.1M Tris-glycine, pH 8.01. The chymotrypsin was eluted from the column using a buffer of 0.1M glycine-HCl at a pH of 2.07. Analysis of the column effluent via UV absorption, showed that 96.2% of the chymotrypsin was eluted with the buffer solution. Thus, Kucera was successful in performing affinity chromatography on bead cellulose with a bound ligand, trypsin inhibitor, that has a molecular weight of 14,000, which attracts a bioaffinant, chymotrypsin, that has a molecular weight of 25,000.
In spite of the success of Kucera and others in performing bioaffinity chromatography with cellulose beads, the prior art cellulose beads are incapable of binding bioaffinity ligands having molecular weights greater than 50,000. In general, only small molecular weight ligands and rigid ligands which do not have molecular recognition sites (i.e., the rigid ligands attract and bind to specific molecular recognition sites on the complementary bioaffinants) have been efficiently used for affinity chromatography. This is due to the high concentration of cellulose in the prior art beads which results in a denser bead structure of low porosity (low accessibility to the internal gel structure) when compared to beads made from materials such as agarose.
Typically, prior art cellulose beads used in bioaffinity chromatography are dried to reduce bead size, and, after drying and reconstitution in water, contain approximately 80% water by weight (20% cellulose by weight). Even at these cellulose concentrations, the beads are still insufficiently porous to allow for their use in bioaffinity chromatography of high molecular weight bioaffinants and bioaffinity ligands. However, a reduction in the amount of cellulose in the beads to make them more porous may compromise the strength of the beads.
Thus, there is a need for low cellulose beads, i.e., having reduced cellulose content, for use in bioaffinity chromatography that are sufficiently porous to allow molecules with molecular weights ranging from 5,000 to 5,000,000 to have access to their interiors; such high porosity, low cellulose beads are needed for use in bioaffinity chromatography of high molecular weight molecules, such as antibodies, therapeutic proteins, and enzymes. There is also a need for a bioaffinity chromatography support which has, in addition to the foregoing desired properties, high mechanical strength to resist crushing at high column flow rates, and which does not suffer from nonspecific adsorption by the support matrix of complex solution components. Further, there is a need for a support for high molecular weight bioaffinity chromatography which is made from an inexpensive and readily accessible material, and which is produced by a simple and inexpensive process that is readily adaptable for large scale production.
Therefore, it is a primary object of this invention to produce beads of cellulose of sufficient porosity to enable its use in bioaffinity chromatography with ligands having molecular weights between 5,000 and 500,000 and bioaffinants having molecular weights between 5,000 and 500,000.
It is a further object of this invention to produce beads of cellulose suitable for bioaffinity chromatography of high molecular weight substances which have high strength, and are inexpensive to produce.
It is yet another object of this invention to provide a process for the production of cellulose beads for use in bioaffinity chromatography having cellulose or lignocellulose present in quantities low enough to permit the beads to be used in bioaffinity chromatography of high molecular weight proteins.
It is a further object of this invention to provide a process for performing bioaffinity chromatography of high molecular weight proteins, such as, but not limited to, antibodies, therapeutic proteins, and enzymes, on bead cellulose.